Exposure and biological response biomarkers of cigarette smoke. Exposure to tobacco smoke (mainstream and environmental) is a leading cause of death in the US. Cigarette smoke is an extremely complex mixture, which some 3800 constituents including numerous polycyclic aromatic hydrocarbons (PAHs), in both the mainstream and sidestream (environmental) smoke fractions. Cigarette smokers provide an extreme model of PAH exposure that will permit both exposure and biological response biomarkers to be developed. There is substantial evidence that PAHs are causative agents in lung, skin, and bladder cancer. Furthermore, tobacco smoke is associated with oxidative stress, pancreatic cancer, cardiovascular disease, and chronic obstructive pulmonary disease (COPD), although the specific role of PAHs is not clear. Interestingly, the cardiovascular effects of sidestream smoke are almost as great as mainstream smoke. The present proposal stems from significant advances we have made over the last six years in the quantification of protein, lipid, and DNA biomarkers using stable isotope methodology and our basic research into enzyme regulation during oxidative stress. Previous methods for analyzing oxidative DNA damage have been fraught with numerous methodological problems so that the current state-of-the-art involves the use of a COMET assay to measure 8-oxo-2'-deoxyguanosine (dGuo) lesions. We have recently devised a more quantitative method based on immunoaffinity stable isotope dilution liquid chromatography- tandem mass spectrometry (LC-MS/MS) that can be readily elaborated to studies of tobacco smokers. We also showed that oxidative stress could induce the formation of aldo-keto reductases (AKRs) of the 1C family. AKR1C3 is the enzyme, which we recently showed is responsible for the conversion of prostaglandin (PG) D2 to the potent bronchoconstrictor 11p-PGF2. This provides an additional potential link between oxidative stress and COPD as well as the potential for a new therapeutic strategy, which involves AKR1C3 inhibition. Finally, preliminary studies have revealed that a DNA-adduct than can only arise from lipid peroxidation is present in the urine of cigarette smokers but is completely absent in urine from non-smokers. We propose to build on these exciting new findings by developing panels of in vivo biomarkers of exposure and biological response, which we hypothesize will make it possible to distinguish a cohort of non-smokers from a cohort of disease-free tobacco smokers. The hypothesis will be tested by conducting research under the following three specific aims: Aim 1. To discover whether B[a]P and B[a]P-7,8-dione induce AKR1C/2 in NHBE cells and increase oxidative stress to form 8-oxo-dGuo and HedGuo in DNA, induce AKR1C3 in HASM cells and increase the biosynthesis of the potent bronchoconstrictor 11p-PGF2, as potential urine and EEC biological response biomarkers of PAH exposure. Aim 2: To discover secreted proteins following treatment of NHBE and HASM cells with B[a]P and its oxidative metabolites as potential serum biological response biomarkers of PAH exposure. Aim 3: To conduct predictive and refinement analyses of in vivo exposure and response biomarkers in urine together with biological response biomarkers in EBC and serum in order to distinguish non-smokers from disease-free tobacco smokers. Successful completion of the proposed research will provide a panel of biomarkers of exposure and biological response to tobacco smoke will have significant utility in future studies designed to elucidate the relationship between gene environment interactions and diseases such as cancer, cardiovascular disease, and COPD.